Sororin mediates sister chromatid cohesion by antagonizing Wapl.

Sister chromatid cohesion is essential for chromosome segregation and is mediated by cohesin bound to DNA. Cohesin-DNA interactions can be reversed by the cohesion-associated protein Wapl, whereas a stably DNA-bound form of cohesin is thought to mediate cohesion. In vertebrates, Sororin is essential for cohesion and stable cohesin-DNA interactions, but how Sororin performs these functions is unknown. We show that DNA replication and cohesin acetylation promote binding of Sororin to cohesin, and that Sororin displaces Wapl from its binding partner Pds5. In the absence of Wapl, Sororin becomes dispensable for cohesion. We propose that Sororin maintains cohesion by inhibiting Wapl's ability to dissociate cohesin from DNA. Sororin has only been identified in vertebrates, but we show that many invertebrate species contain Sororin-related proteins, and that one of these, Dalmatian, is essential for cohesion in Drosophila. The mechanism we describe here may therefore be widely conserved among different species.

The Value of Torque Teno Virus (TTV) as a Marker for the Degree of Immunosuppression in Adult Patients after Hematopoietic Stem Cell Transplantation (HSCT).

Torque teno virus (TTV) is a nonenveloped, single-stranded, circular DNA virus of the family of Anelloviridae. The first contact with TTV usually occurs in early childhood, followed by persistent infection in bone marrow and lymphocytes. Increased levels of TTV-DNA are found in the serum in various states of immune deficiency. The objective of this study was to assess if monitoring of TTV viremia after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a predictive marker for immune-related clinical complications. In a retrospective study, 2054 whole-blood samples from 123 patients were tested for viral loads of TTV-DNA by real-time PCR within 345 days after allo-HSCT. We enrolled all patients who underwent allo-HSCT between September 2015 and April 2018. Clinical and laboratory data were collected and statistically analyzed. Patients with an underlying lymphatic malignancy had significantly higher torque teno (TT) viral loads compared with patients with an underlying malignant myeloid disease (P < .05). Complete remission before allo-HSCT correlated significantly with higher TT viral loads after allo-HSCT (P < .05). Myeloablative conditioning regimens led to significantly higher TT viral loads than reduced-intensity conditioning regimens (P < .05). A higher anti-thymocyte globulin (ATG) dose was associated with a significantly higher TT viral load. We did not observe any significant differences of TT viral load correlating with accompanying clinically relevant events such as virus reactivations (cytomegalovirus, Epstein-Barr virus, Adenovirus), acute graft-versus-host disease (aGVHD), relapse, or death. TT viral load after allo-HSCT did weakly correlate with T cell, T suppressor cell, T helper cell, and natural killer and B cell count. Although statistically significant differences between study groups were observed, virus reactivations, aGVHD, and clinical outcomes could not be predicted by monitoring TTV viremia. Therefore, TTV seems not to be suitable as a marker for the degree of immunosuppression or as a prognostic marker for clinically critical events in patients after allo-HSCT.

Positive Treatment Expectancies Reduce Clinical Pain and Perceived Limitations in Movement Ability Despite Increased Experimental Pain: A Randomized Controlled Trial on Sham Opioid Infusion in Patients with Chronic Back Pain.

BACKGROUND: Increasing evidence for the efficacy of analgesic placebo effects in laboratory studies with healthy persons raises the question whether placebos could be used to improve the treatment of pain patients. Expectancies play a central role in shaping analgesic placebo but also nocebo effects. OBJECTIVES: We investigated to what extent a sham opioid infusion (saline solution) produces sustained clinically relevant placebo and nocebo effects in chronic back pain patients. METHODS: Fifty-nine patients received the sham opioid infusion applied via a large drain dressing and were compared to 14 control patients without intervention (natural history, NH) while experimental pain stimuli were applied. All subjects were told that the infusion would decrease pain although in rare cases pain increase would be possible (induction of expectancy). In addition, conditioning was introduced where the participants either experienced a decrease in experimental pain (n = 17; placebo conditioning), an increase (n = 21; nocebo conditioning), or no change (n = 21, no conditioning). RESULTS: Compared to the NH group, all infusion groups showed positive treatment expectancies and significantly (p < 0.001) reduced clinical back pain (primary outcome) and pain-related disability (secondary outcome, assessed by self-reported functional capacity and perceived impairment of mobility). Even the nocebo conditioned group experiencing increased experimental pain developed positive treatment expectancies followed by reduced pain experience. Positive treatment expectancies and relief in clinical back pain were significantly positively correlated (r = 0.72, p < 0.01). CONCLUSIONS: These findings suggest that it may be beneficial to explicitly shape and integrate treatment expectancies into clinical pain management.

SNW1 enables sister chromatid cohesion by mediating the splicing of sororin and APC2 pre-mRNAs.

Although splicing is essential for the expression of most eukaryotic genes, inactivation of splicing factors causes specific defects in mitosis. The molecular cause of this defect is unknown. Here, we show that the spliceosome subunits SNW1 and PRPF8 are essential for sister chromatid cohesion in human cells. A transcriptome-wide analysis revealed that SNW1 or PRPF8 depletion affects the splicing of specific introns in a subset of pre-mRNAs, including pre-mRNAs encoding the cohesion protein sororin and the APC/C subunit APC2. SNW1 depletion causes cohesion defects predominantly by reducing sororin levels, which causes destabilisation of cohesin on DNA. SNW1 depletion also reduces APC/C activity and contributes to cohesion defects indirectly by delaying mitosis and causing "cohesion fatigue". Simultaneous expression of sororin and APC2 from intron-less cDNAs restores cohesion in SNW1-depleted cells. These results indicate that the spliceosome is required for mitosis because it enables expression of genes essential for cohesion. Our transcriptome-wide identification of retained introns in SNW1- and PRPF8-depleted cells may help to understand the aetiology of diseases associated with splicing defects, such as retinosa pigmentosum and cancer.

Fluoroquinolone resistance in Streptococcus pneumoniae isolates in Germany from 2004-2005 to 2014-2015.

Streptococcus pneumoniae is a major cause of bacterial pneumonia, sepsis and meningitis worldwide. Prevalence of levofloxacin-resistant S. pneumoniae isolates in Germany and associated mutations in the quinolone resistance determining regions (QRDRs), as well as serotype distribution and multi locus sequence types (MLST) are shown. 21,764 invasive S. pneumoniae isolates from Germany, isolated in the epidemiological seasons from 2004/05 to 2014/15 were analyzed at the German National Reference Centre for Streptococci (GNRCS) for their levofloxacin resistance by micro broth dilution method. All resistant (minimal inhibitory concentration (MIC) ≥8µg/ml) and intermediate (MIC >2µg/ml and <8µg/ml) isolates were selected for the present study. Additionally, 29 susceptible isolates were randomly selected. A total of ninety isolates were tested for their levofloxacin-MIC by Etest, their serotype and sequence type, as well as for point-mutations at the QRDRs in the genes parC, parE, gyrA and gyrB. Twenty-five isolates exhibited levofloxacin MICs <2µg/ml (Etest) and no mutations in the QRDRs. Four isolates with MICs=2µg/ml had one mutation in parC; isolates with MICs >2µg/ml all had one or more mutations in the QRDRs. Four of nine intermediate isolates had a mutation in either parC or gyrA, and four isolates had mutations in both parC and gyrB. One isolate had mutations in both parC and gyrA. All isolates with MICs ≥8µg/ml (52) had mutations in both topoisomerase IV and gyrase. Serotypes associated with levofloxacin resistance shifted from a majority of PCV13 serotypes before the introduction of the PCV13 vaccine towards non-PCV serotypes. Resistant isolates were almost exclusively found among adults (98.1%).

Sororin is required for stable binding of cohesin to chromatin and for sister chromatid cohesion in interphase.

Sister chromatid cohesion depends on cohesin [1-3]. Cohesin associates with chromatin dynamically throughout interphase [4]. During DNA replication, cohesin establishes cohesion [5], and this process coincides with the generation of a cohesin subpopulation that is more stably bound to chromatin [4]. In mitosis, cohesin is removed from chromosomes, enabling sister chromatid separation [6]. How cohesin associates with chromatin and establishes cohesion is poorly understood. By searching for proteins that are associated with chromatin-bound cohesin, we have identified sororin, a protein that was known to be required for cohesion [7]. To obtain further insight into sororin's function, we have addressed when during the cell cycle sororin is required for cohesion. We show that sororin is dispensable for the association of cohesin with chromatin but that sororin is essential for proper cohesion during G2 phase. Like cohesin, sororin is also needed for efficient repair of DNA double-strand breaks in G2. Finally, sororin is required for the presence of normal amounts of the stably chromatin-bound cohesin population in G2. Our data indicate that sororin interacts with chromatin-bound cohesin and functions during the establishment or maintenance of cohesion in S or G2 phase, respectively.

Alpha4beta1-dependent adhesion strengthening under mechanical strain is regulated by paxillin association with the alpha4-cytoplasmic domain.

The capacity of integrins to mediate adhesiveness is modulated by their cytoplasmic associations. In this study, we describe a novel mechanism by which alpha4-integrin adhesiveness is regulated by the cytoskeletal adaptor paxillin. A mutation of the alpha4 tail that disrupts paxillin binding, alpha4(Y991A), reduced talin association to the alpha4beta1 heterodimer, impaired integrin anchorage to the cytoskeleton, and suppressed alpha4beta1-dependent capture and adhesion strengthening of Jurkat T cells to VCAM-1 under shear stress. The mutant retained intrinsic avidity to soluble or bead-immobilized VCAM-1, supported normal cell spreading at short-lived contacts, had normal alpha4-microvillar distribution, and responded to inside-out signals. This is the first demonstration that cytoskeletal anchorage of an integrin enhances the mechanical stability of its adhesive bonds under strain and, thereby, promotes its ability to mediate leukocyte adhesion under physiological shear stress conditions.

Helicobacter pylori gastritis in children is associated with a regulatory T-cell response.

BACKGROUND & AIMS: Helicobacter pylori infection in children infrequently causes gastroduodenal mucosal ulceration. Because H pylori induces T-cell dependent gastric inflammation in adults and T regulatory (Treg) cells suppress T-cell-dependent pathology, we evaluated gastric histopathology and Treg cell responses in H pylori-infected children and adults. METHODS: Gastric tissue from 36 children and 79 adults with abdominal symptoms in Santiago, Chile, was evaluated prospectively for H pylori bacteria and histopathology using the Sydney classification and Treg responses using immunoassay, immunohistochemistry, and real-time polymerase chain reaction. RESULTS: Eighteen (50%) of the children and 51 (65%) of the adults were infected with H pylori. Children and adults were colonized with similar levels of H pylori. However, the level of gastritis in the children was reduced substantially compared with that of the adults (P < .05). Coincident with reduced gastric inflammation, the number of Treg cells and levels of Treg cytokines (transforming growth factor [TGF]-beta1 and interleukin-10) were increased markedly in the gastric mucosa of H pylori-infected children compared with that of infected adults (P < .03 and < .05, respectively). Also, H pylori infection in the children was associated with markedly increased levels of gastric TGF-beta1 and interleukin-10 messenger RNA. Importantly, gastric TGF-beta1 in H pylori-infected children localized predominantly to mucosal CD25(+) and Foxp3(+) cells, indicating a Treg source for the TGF-beta1. CONCLUSIONS: Gastric pathology is reduced and local Treg cell responses are increased in H pylori-infected children compared with infected adults, suggesting that gastric Treg cell responses down-regulate the inflammation and ulceration induced by H pylori in children.

Gastric mucus alterations associated with murine Helicobacter infection.

The C57BL/6 mouse has been shown to develop gastric adenocarcinoma after Helicobacter felis infection. This model was used to determine whether mucin and trefoil factor (TFF) expression after infection was altered in a similar fashion to the changes seen in the protective gastric mucus layer of the human stomach after H. pylori infection. Our results indicate that this mouse model mimics many of the changes seen after human H. pylori infection, including increased expression of muc4 and muc5b and loss of muc5ac. These alterations in mucin expression occurred as early as 4 weeks postinfection, before the development of significant mucous metaplasia or gastric dysplasia. The decrease in muc5ac expression occurred only in the body of the stomach and was not secondary to the adaptive immune response to infection, because a similar decrease in expression was seen after infection of B6.Rag-1(-/-) mice, which lack B and T cells. Intriguingly, the increased expression of Muc4 and Muc5b in infected C57BL/6 mice was not seen in the infected B6.Rag-1(-/-) mice. Because B6.Rag-1(-/-) mice do not develop gastric pathology after H. felis infection, these findings point to the potential role of Muc4 and Muc5b in disease progression. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

A Low Tacrolimus Concentration/Dose Ratio Increases the Risk for the Development of Acute Calcineurin Inhibitor-Induced Nephrotoxicity.

Fast tacrolimus metabolism is linked to inferior outcomes such as rejection and lower renal function after kidney transplantation. Renal calcineurin-inhibitor toxicity is a common adverse effect of tacrolimus therapy. The present contribution hypothesized that tacrolimus-induced nephrotoxicity is related to a low concentration/dose (C/D) ratio. We analyzed renal tubular epithelial cell cultures and 55 consecutive kidney transplant biopsy samples with tacrolimus-induced toxicity, the C/D ratio, C0, C2, and C4 Tac levels, pulse wave velocity analyses, and sublingual endothelial glycocalyx dimensions in the selected kidney transplant patients. A low C/D ratio (C/D ratio < 1.05 ng/mL×1/mg) was linked with higher C2 tacrolimus blood concentrations (19.2 ± 8.7 µg/L vs. 12.2 ± 5.2 µg/L respectively; p = 0.001) and higher degrees of nephrotoxicity despite comparable trough levels (6.3 ± 2.4 µg/L vs. 6.6 ± 2.2 µg/L respectively; p = 0.669). However, the tacrolimus metabolism rate did not affect the pulse wave velocity or glycocalyx in patients. In renal tubular epithelial cells exposed to tacrolimus according to a fast metabolism pharmacokinetic profile it led to reduced viability and increased Fn14 expression. We conclude from our data that the C/D ratio may be an appropriate tool for identifying patients at risk of developing calcineurin-inhibitor toxicity.

Murine Adherent and Invasive E. coli Induces Chronic Inflammation and Immune Responses in the Small and Large Intestines of Monoassociated IL-10-/- Mice Independent of Long Polar Fimbriae Adhesin A.

BACKGROUND: Adherent and invasive Escherichia coli (AIEC) is preferentially associated with ileal Crohn's disease (CD). The role of AIEC in the development of inflammation and its regional tropism is unresolved. The presence of long polar fimbriae (LPF) in 71% of ileal CD AIEC suggests a role for LPF in the tropism and virulence of AIEC. The aim of our study is to determine if AIEC, with or without LpfA, induces intestinal inflammation in monoassociated IL-10-/- mice. METHODS: We compared murine AIEC strains NC101 (phylogroup B2, LpfA-) and CUMT8 (phylogroup B1, LpfA+), and isogenic mutant CUMT8 lacking lpfA154, with a non-AIEC (E. coli K12), evaluating histologic inflammation, bacterial colonization, mucosal adherence and invasion, and immune activation. RESULTS: IL-10-/- mice monoassociated with AIEC (either CUMT8, CUMT8:ΔlpfA, or NC101) but not K12 developed diffuse small intestinal and colonic inflammation. There was no difference in the magnitude and distribution of inflammation in mice colonized with CUMT8:ΔlpfA compared with wild-type CUMT8. Bacterial colonization was similar for all E. coli strains. Fluorescence in situ hybridization revealed mucosal adherence and tissue invasion by AIEC but not K12. Production of the cytokines IL-12/23 p40 by the intestinal tissue and IFN-γ and IL-17 by CD4 T cells correlated with inflammation. CONCLUSIONS: IL-10-/- mice monoassociated with murine AIEC irrespective of LpfA expression developed chronic inflammation accompanied by IL-12/23 p40 production in the small and large intestines and IFN-γ/IL-17 production by CD4 T cells that model the interplay between enteric pathosymbionts, host susceptibility, and enhanced immune responses in people with IBD.

The viscoelasticity of membrane tethers and its importance for cell adhesion.

Cell adhesion mechanically couples cells to surfaces. The durability of individual bonds between the adhesive receptors and their ligands in the presence of forces determines the cellular adhesion strength. For adhesive receptors such as integrins, it is a common paradigm that the cell regulates its adhesion strength by altering the affinity state of the receptors. However, the probability distribution of rupture forces is dependent not only on the affinity of individual receptor-ligand bonds but also on the mechanical compliance of the cellular anchorage of the receptor. Hence, by altering the anchorage, the cell can regulate its adhesion strength without changing the affinity of the receptor. Here, we analyze the anchorage of the integrin VLA-4 with its ligand VCAM-1. For this purpose, we develop a model based on the Kelvin body, which allows one to quantify the mechanical properties of the adhesive receptor's anchorage using atomic force microscopy on living cells. As we demonstrate, the measured force curves give valuable insight into the mechanics of the cellular anchorage of the receptor, which is described by the tether stiffness, the membrane rigidity, and the membrane viscosity. The measurements relate to a tether stiffness of k(t) = 1.6 microN/m, an initial membrane rigidity of k(i) = 260 microN/m, and a viscosity of mu = 5.9 microN x s/m. Integrins exist in different activation states. When activating the integrin with Mg(2+), we observe altered viscoelastic parameters of k(t) = 0.9 microN/m, k(i) = 190 microN/m, and mu = 6.0 microN x s/m. Based on our model, we postulate that anchorage-related effects are common regulating mechanisms for cellular adhesion beyond affinity regulation.

Mechanical regulation of cell adhesion.

Cellular adhesion against external forces is governed by both the equilibrium affinity of the involved receptor-ligand bonds and the mechanics of the cell. Certain receptors like integrins change their affinity as well as the mechanics of their anchorage to tune the adhesiveness. Whereas in the last few years the focus of integrin research has lain on the affinity regulation of the adhesion receptors, more recently the importance of cellular mechanics became apparent. Here, we focus on different aspects of the mechanical regulation of the cellular adhesiveness.

Clinical Use of Placebo Effects in Patients With Pain Disorders.

The analgesic placebo effect is well documented by numerous studies. Many important influencing factors, however, are yet to be discovered. In the arena of placebo effects and clinical implications, expectancies play a central role. Expectancies are shaped by processes of classical and social learning as well as verbal instructions and are strongly related to emotional factors. Expectancies trigger a cascade of endogenous opioids and non-opioids, which alter the experience of pain. For clinical application it is important to know, that placebo research yields ethical possibilities to use placebo effects without deception and without using placebos. Since placebo effects contribute to responses to active analgesics, it is feasible to enhance patients' benefits from pain treatments by increasing the additional placebo effect. There are several possibilities to use the placebo effects via shaping and adapting information about analgesic medication and via associating medication intake with a positive context. A positive patient-clinician communication atmosphere is very important to generate clinically meaningful placebo effects in pain medicine.

Expression of CXCL15 (Lungkine) in murine gastrointestinal, urogenital, and endocrine organs.

The ELR(+) chemokine CXCL15, which recruits neutrophils during pulmonary inflammation, is also known as lungkine due to its reported exclusive expression in the lung. We now report that CXCL15 mRNA and protein are also expressed in other mucosal and endocrine organs including the gastrointestinal and urogenital tracts and the adrenal gland. Our results indicate that CXCL15 is expressed throughout the gastrointestinal tract, with the exception of the cecum. Gastric CXCL15 protein expression is approximately 10-fold lower than pulmonary expression and primarily occurs in a specific lineage of gastric epithelial cells, the prezymogenic and zymogenic cell. Similar to the increased expression of CXCL15 during pulmonary inflammation, gastric inflammation induced by infection with Helicobacter felis caused an increase in gastric CXCL15 expression. However, colonic CXCL15 expression was not altered in two different models of colonic inflammation, the Helicobacter hepaticus T-cell transfer model and the mdr1a(-/-) model of colitis. These findings clearly demonstrate that CXCL15, previously reported to be the only lung-specific chemokine, is also highly expressed in other murine mucosal and endocrine organs. The functional role of CXCL15 in mucosal disease remains to be elucidated. This manuscript contains online supplemental material at (http://www.jhc.org). Please visit this article online to view these materials.

Nocebo effects in clinical studies: hints for pain therapy.

INTRODUCTION: Nocebo-induced algesic responses occurring within clinical contexts present a challenge for health care practitioners working in the field of pain medicine. OBJECTIVES: Following the recent research on algesic nocebo effects, the scope of this review is to develop ethically acceptable strategies to help avoid, or at least reduce, nocebo responses within clinical settings. METHODS: We reviewed relevant clinical studies that depict how patient-practitioner interactions may contribute to the reduction of nocebo responses. RESULTS: A strong algesic nocebo effect may adversely impact a patient's condition by causing decreases in both the efficacy and effectiveness of interventions, as well as by promoting treatment nonadherence and discontinuation. These effects may be triggered through multiple channels and can lead to significant alterations in a patient's perception of pain, consequently producing a weakening of the specific positive effects of pharmacological, psychological, or physical pain-management interventions. CONCLUSION: To minimize nocebo effects in clinical settings, we identified and discussed five contextual aspects relevant to the treatment of patients with chronic pain: (1) negative patient-clinician communication and interaction during treatment; (2) emotional burden of patients during treatment with analgesic medication; (3) negative information provided via informational leaflets; (4) cued and contextual conditioning nocebo effects; and (5) patient's lack of positive information. Through an understanding of these elements, many preventive and ethically acceptable clinical actions can be taken to improve multidisciplinary pain treatment outcomes.

Placebo effects of a sham opioid solution: a randomized controlled study in patients with chronic low back pain.

This study tested the experimental placebo effect in a group of chronic pain patients. Forty-eight patients having chronic back pain participated in a randomized clinical trial that tested the efficacy of a sham opioid solution (NaCl) compared with an alleged neutral, completely inactive solution (NaCl). We shaped the placebo effect by 2 interventions: verbal instruction and conditioning. The patients were either told that the "solution reduces pain and improves physical capacity" or the "solution is neutral, a placebo." Half of each group was additionally conditioned (coupling solution with reduced experimental pain), yielding 4 subgroups with 12 participants each. Outcome measures were as follows: the patients' clinical back pain ratings and acute pain ratings (both examined by numerical rating scale 0-10) and self-rated functional capacity (0%-100%; time required for the exercise). Expected pain relief before and after solution intake was also assessed. The inactive solution (NaCl), when presented as an effective treatment (sham "opioid" solution), induced placebo analgesia as evident in lower ratings of the patients' clinical back pain (F(3.12,144.21) = 25.05, P < 0.001), acute pain ratings (F(1.99,87.40) = 18.12, P < 0.01), and time needed to complete a series of daily activities exercises (F(1,44) = 8.51, P < 0.01) as well as increased functional capacity (F(1,44.00) = 19.42, P < 0.001). The 2 manipulations (instruction and conditioning) changed pain expectations, and they were maintained in both sham opioid groups. The results suggest that it may be clinically useful to explicitly integrate placebo analgesia responses into pain management.

Shiga Toxin Glycosphingolipid Receptors in Human Caco-2 and HCT-8 Colon Epithelial Cell Lines.

Shiga toxins (Stxs) released by enterohemorrhagic Escherichia coli (EHEC) into the human colon are the causative agents for fatal outcome of EHEC infections. Colon epithelial Caco-2 and HCT-8 cells are widely used for investigating Stx-mediated intestinal cytotoxicity. Only limited data are available regarding precise structures of their Stx receptor glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), and lipid raft association. In this study we identified Gb3Cer and Gb4Cer lipoforms of serum-free cultivated Caco-2 and HCT-8 cells, chiefly harboring ceramide moieties composed of sphingosine (d18:1) and C16:0, C22:0 or C24:0/C24:1 fatty acid. The most significant difference between the two cell lines was the prevalence of Gb3Cer with C16 fatty acid in HCT-8 and Gb4Cer with C22-C24 fatty acids in Caco-2 cells. Lipid compositional analysis of detergent-resistant membranes (DRMs), which were used as lipid raft-equivalents, indicated slightly higher relative content of Stx receptor Gb3Cer in DRMs of HCT-8 cells when compared to Caco-2 cells. Cytotoxicity assays revealed substantial sensitivity towards Stx2a for both cell lines, evidencing little higher susceptibility of Caco-2 cells versus HCT-8 cells. Collectively, Caco-2 and HCT-8 cells express a plethora of different receptor lipoforms and are susceptible towards Stx2a exhibiting somewhat lower sensitivity when compared to Vero cells.